Various types of RNAs are expressed in vivo. In addition to mRNAs encoding amino acid sequences of proteins, a large amount of RNAs encoding non-translatable regions, known as non-coding RNAs, are found. Among non-coding RNAs, micro RNAs (miRNAs) are small RNAs of 18 to 25 nucleotides present in cells. Micro RNAs play important regulatory roles in development of animals, plants, and viruses. Further, recent studies have revealed that miRNAs have regulatory functions in translation of proteins in animal cells. Studies on miRNAs have dramatically advanced since then, and 24,521 miRNAs are now registered in the miRBase database version 19 (http://www.mirbase.org/), and 25,646 articles related to miRNAs are included in the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed). These reflect the high level of interest in miRNAs and the importance of miRNAs.
Micro RNAs regulate and inhibit translation by binding to target messenger RNAs. Presumably, miRNAs regulate transcription and expression of about 30% of the human genome.
Moreover, regarding the importance of miRNAs, it has been demonstrated that miRNAs are involved in development, growth, proliferation, apoptosis, differentiation, and various cellular expression processes, including various human diseases such as cancer and diabetes.
Non-Patent Literature 1 emphasizes the significance of miRNAs in cancer and describes involvement of miRNAs in various types of human cancer. Non-Patent Literature 1 states that measuring changes in the expression level of miRNAs is greatly useful for advancing cancer research.
Conventional methods for detecting miRNAs include qRT-PCR and microarray assay. However, since miRNAs are small RNAs each consisting of 18 to 25 nucleotides, they have been very difficult to amplify or detect. Non-Patent Literature 2, Patent Literature 1, and Non-Patent Literature 3 have suggested solutions to this problem. Non-Patent Literature 2 discloses a qRT-PCR assay including a gene-specific reverse transcription step and a subsequent fluorescent detection step using a gene-specific forward primer with TaqMan probe and a universal reverse primer. Also in Patent Literature 1, miRNAs are detected by fluorescence using similar reverse transcription reaction primers. Non-Patent Literature 3 discloses a qRT-PCR assay including a gene-specific reverse transcription step and a subsequent SYBR (registered trademark) green qPCR step using a gene-specific forward primer and two universal primers.
However, there is no known method for detecting miRNAs by qRT-PCR or carrying out microarray assay in a rapid and simple manner.